Anti rabbit secondary antibodies

COS (average molecular weight ≈ 1000, degree of polymerization 3–7, purity 97%) are provided by the Yuan Ye Biology (Shanghai, China). DMEM and DPBS were purchased from HyClone (Logan, UT, USA), and fetal bovine serum (FBS) was purchased from Corning (Manassas, VA, USA). CCK8 kit was purchased from Zeta Life (Menlo Park, CA, USA), the Edu cell proliferation assay kit was purchased from RiboBio (Guangzhou, Guangdong, China), and Cell Cycle and Apoptosis Analysis Kit were purchased from Beyotime (Shanghai, China). The triglyceride and cholesterol detection kit was purchased from Pulilai (Beijing, China). The non-esterified fatty acids assay kit was purchased from Nanjing Jian cheng Bioengineering Institute (Nanjing, Jiangsu, China). The primary antibodies against AMPKα1, SREBP1, SCD1 and FASN were purchased from Bioss (Beijing, China). The antibodies against HSL were purchased from Cell Signaling Technology (Boston, MA, USA). The antibodies against β-actin and Anti-rabbit secondary antibodies were purchased from Bioworld Technology CO., Ltd. (Minneapolis, MN, USA).

Protein concentrations in the cell lysates were determined by Nanodrop ND-1000 (Gene Co., Ltd., Hong Kong, china). Once protein concentrations were determined, 50 µl of the protein specimens were denatured in 50 µl of 2×sample buffer (125 mmol/L Tris-HCl, pH 6.8, 20% glycerol, 10% β-mercaptoethanol, 0.02% bromophenol blue, and 4% SDS). Briefly, 30 µg of denatured protein were separated using a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinyl difluoride (PVDF) membrane, which was blocked in 5% milk proteins that were suspended in tris-buffered saline tween-20 (TBST) for 2 h at room temperature. The membrane was rinsed three times with TBST followed by incubation with the primary antibody (Cell Signaling Technology, Inc, USA) overnight at 4°C. The membrane was then washed and treated with anti-rabbit secondary antibodies that were conjugated with horseradish peroxidase at a 1∶5000 dilution (Bioworld Technology Inc, USA). The immunoreactive proteins were visualized with a chemiluminescence detection kit (PerkinElmer, USA), and the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein was used as the loading control across all samples analyzed by Western blot.

DMEM and DPBS were purchased from HyClone (UT, USA). Fetal bovine serum (FBS) was purchased from ZATA Life (CA, USA). The triglycerides and cholesterol detection kit were purchased from Pulilai (Beijing, China). The non-esterified fatty acids assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The lipid dye BODIPY505/515 was purchased from Invitrogen (Waltham, MA, USA), and three ELISA kits (IL-6, IL-1β and TNF-α) were purchased from MEIMIAN (Wuxi, China). The primary antibodies against AMPKα1, Phosphorylated AMP-activated protein kinase (p-AMPK), SDC3, PPARG, and Phospho-IκB Alpha (p-IκBα) were purchased from Bioss (Beijing, China). Antibodies against nuclear factor kappa B subunit 1 (NF-κB p50), IκBα, and SIRT1 were purchased from Proteintech Group (Wuhan, China). The antibodies against β-actin and anti-rabbit secondary antibodies were acquired from Bioworld Technology (St. Louis Park, MN, USA).